Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope

Sigler, Albrecht; Goroshkov, Alexander V.; Murphy, Timothy H.

doi:10.1016/j.jneumeth.2007.12.015

Cited by 52 publications

(58 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The procedures for laser speckle imaging have been described previously (Dunn et al, 2001;Sigler et al, 2008). Briefly, the cortical surface was illuminated by a 784 nm, 32 mW StockerYale SNF-XXX-785S-35 laser (StockerYale) with an Edmund Optics anamorphic beam expander T47274 (Edmund Optics), and 50 -150 images at 30 Hz using 10 -15 ms exposure were collected with a Dalsa M60 Pantera camera (Dalsa).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Two-Photon Imaging during Prolonged Middle Cerebral Artery Occlusion in Mice Reveals Recovery of Dendritic Structure after Reperfusion

Li¹,

Murphy²

2008

J. Neurosci.

115

View full text Add to dashboard Cite

Filament occlusion of the middle cerebral artery (MCA) is a well accepted animal model of focal ischemia. Advantages of the model are relatively long occlusion times and a large penumbra region that simulates aspects of human stroke. Here, we use two-photon and confocal microscopy in combination with regional measurement of blood flow using laser speckle to assess the spatial relationship between the borders of the MCA ischemic territory and loss of dendrite structure, as well as the effect of reperfusion on dendritic damage in adult YFP (yellow fluorescent protein) and GFP (green fluorescent protein) C57BL/6 transgenic mice with fluorescent (predominantly layer 5) neurons. By examining the spatial extent of dendritic damage, we determined that 60 min of MCA occlusion produced a core with severe structural damage that did not recover after reperfusion (begins ϳ3.8 mm lateral to midline), a reversibly damaged area up to 0.6 mm medial to the core that recovered after reperfusion (penumbra), and a relatively structurally intact area (ϳ1 mm wide; medial penumbra) with hypoperfusion. Loss of structure was preceded by a single ischemic depolarization 122.1 Ϯ 10.2 s after occlusion onset. Reperfusion of animals after 60 min of ischemia was not associated with exacerbation of damage (reperfusion injury) and resulted in a significant restoration of blebbed dendritic structure, but only within ϳ0.6 mm lateral of the dendritic damage structural border. In summary, we find that recovery of dendritic structure can occur after reperfusion after even 60 min of ischemia, but is likely restricted to a relatively small penumbra region with partial blood flow or oxygenation.

show abstract

Section: Methodsmentioning

confidence: 99%

“…5C) and could account for some of the spared dendrites. Across the laser speckle image, a gradual darkening is apparent toward the medial portion of the image and is attributed to diffusion in microvessels (Dunn et al, 2001;Yuan et al, 2005;Sigler et al, 2008), indicating higher levels of local blood flow.…”

Section: Blood Flow and Structural Change During Mcaomentioning

confidence: 99%

Two-Photon Imaging during Prolonged Middle Cerebral Artery Occlusion in Mice Reveals Recovery of Dendritic Structure after Reperfusion

Li¹,

Murphy²

2008

J. Neurosci.

115

View full text Add to dashboard Cite

show abstract

“…Although laser-mediated excitation was still used recently5, it can be replaced by cold light lamp that also ensure dye excitation 10,15 . Cold light optic fibers are easier to manipulate and less expensive than laser sources.…”

Section: Modifications and Substitutionsmentioning

confidence: 99%

“…Cold light optic fibers are easier to manipulate and less expensive than laser sources. However, it should be noticed that lasers are commonly used to target individual surface arterioles after craniotomy for an in vivo vessel-specific clotting 10 .…”

Section: Modifications and Substitutionsmentioning

confidence: 99%

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

Labat-Gest

Tomasi

2013

JoVE

173

143

View full text Add to dashboard Cite

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice. Video LinkThe video component of this article can be found at

show abstract

“…We have recently used it to examine the relationship between changes in blood flow and changes in neuronal structure and function with a modified setup. 30 The work by Zhang and Murphy 26 combined structural assessment of neurons using yellow fluorescent protein fluorescence with 2-photon analysis of blood flow as well as functional measures, including intrinsic optical signals, that defined the range with which individual blood vessels maintain synaptic structure and function, discussed in more detail subsequently.…”

Section: Fluorescent Dextran Imaging Of Vascular Structure and Functimentioning

confidence: 99%

In Vivo 2-Photon Imaging of Fine Structure in the Rodent Brain

Sigler

Murphy

2010

Stroke

Self Cite

View full text Add to dashboard Cite

Abstract-The recent application of 2-photon microscopy to biological specimens has allowed investigators to examine individual synapses within live animals. The gain in resolution over conventional in vivo imaging techniques has been several orders of magnitude. We outline steps for the preparation and maintenance of animals for 2-photon microscopy of fine brain structure. We discuss the in vivo resolution of the method and the ability to image blood flow and synaptic structure in vivo. Applications of in vivo 2-photon microscopy include the study of synapse turnover in adult animals under normal conditions and during pathology such as stroke. In the case of stroke, 2-photon imaging has revealed marked swelling of dendrites and loss of spines within minutes of ischemic onset. Surprisingly, restoration of blood flow during reperfusion was associated with a return of relatively normal structure. Over longer time scales, 2-photon imaging revealed elevated rates of synaptogenesis within peri-infarct tissues recovering from stroke. These results provide an example of how high-resolution in vivo microscopy can be used to provide insight into both the acute pathology and recovery from stroke damage. (Stroke. 2010;41[suppl 1]:S117-S123.)Key Words: 2-photon microscopy Ⅲ anatomy Ⅲ blood flow Ⅲ brain recovery Ⅲ dendritic spine morphology Ⅲ excitotoxicity Ⅲ fluorescent proteins Ⅲ focal ischemia Ⅲ imaging Ⅲ microscopy Ⅲ mouse model Ⅲ neuropathology

show abstract

Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope

Cited by 52 publications

References 16 publications

Two-Photon Imaging during Prolonged Middle Cerebral Artery Occlusion in Mice Reveals Recovery of Dendritic Structure after Reperfusion

Two-Photon Imaging during Prolonged Middle Cerebral Artery Occlusion in Mice Reveals Recovery of Dendritic Structure after Reperfusion

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

In Vivo 2-Photon Imaging of Fine Structure in the Rodent Brain

Contact Info

Product

Resources

About